Project II is designed to discover the molecular basis of CFTR-mediated restraint of ENaC activity in human airway epithelia. CFTR-mediated chloride secretion and ENaC-mediated sodium absorption have been considered central to control of airway surface liquid hydration, but the concept of reciprocal control of these opposing ion transport pathways is controversial. In some tissues where CFTR and ENaC are each expressed, their stimulation is coordinated to achieve Na and Cl absorption. Indeed, that is the case in alveolar type 2 cells (AT2C), which are to be characterized in Project III of this PPG. However, in airway epithelia, abundant evidence suggests that CFTR restrains ENaC function. We recently reported that CFTR and ENaC coimmunoprecipitate in normal airway epithelia, and ENaC in CF bronchial epithelium undergoes more extensive cleavage than ENaC in normal. Because ENaC mediated Na+ absorption is activated by cleavage of ENaC extracellular domains, this finding is congruent with the notion that CFTR restrains ENaC cleavage in normal bronchial epithelia. Project II proposes to identify the molecular basis of CFTR-ENaC associations in airway epithelia, which will be accomplished by biochemical assays, comparisons of CFTR and ENaC associated proteins in bronchial airway cells and AT2C, and molecular modeling of detected interactions (Aim 1). With this information. Project II will test two mechanistic hypotheses that describe inhibition of ENaC cleavage. In Aim 2, we hypothesize that physical interaction of the R-domain of CFTR with the cytosolic N-termini of ENaC inhibits ENaC proteolysis and activation. This hypothesis is based on the novel observation that ENaC proteolysis is stimulated by phosphoinositde-binding of its cytosolic N terminal tails. In Aim 3, Project II will test the requirement for CFTR function in the regulation of ENaC cleavage, and will further test specifically how CFTR's control of ASL pH mediates CFTR-specific effects on ENaC proteolysis. Information from these aims will further our understanding of CFTR regulation of ENaC function, specifically addressing the questions of why this regulation is cell specific, and yet when present, how it may be mediated by multiple mechanisms.